Approach could lower cost and eliminate need for antibiotics during biofuel production.

The cost and environmental impact of producing liquid biofuels and biochemicals as alternatives to petroleum-based products could be significantly reduced, thanks to a new metabolic engineering technique.

Liquid biofuels are increasingly used around the world, either as a direct “drop-in” replacement for gasoline, or as an additive that helps reduce carbon emissions.

The fuels and chemicals are often produced using microbes to convert sugars from corn, sugar cane, or cellulosic plant mass into products such as ethanol and other chemicals, by fermentation. However, this process can be expensive, and developers have struggled to cost-effectively ramp up production of advanced biofuels to large-scale manufacturing levels.

One particular problem facing producers is the contamination of fermentation vessels with other, unwanted microbes. These invaders can outcompete the producer microbes for nutrients, reducing yield and productivity.

Ethanol is known to be toxic to most microorganisms other than the yeast used to produce it,Saccharomyces cerevisiae, naturally preventing contamination of the fermentation process. However, this is not the case for the more advanced biofuels and biochemicals under development.

To kill off invading microbes, companies must instead use either steam sterilization, which requires fermentation vessels to be built from expensive stainless steels, or costly antibiotics. Exposing large numbers of bacteria to these drugs encourages the appearance of tolerant bacterial strains, which can contribute to the growing global problem of antibiotic resistance.

Now, in a paper published today in the journal Science, researchers at MIT and the Cambridge startup Novogy describe a new technique that gives producer microbes the upper hand against unwanted invaders, eliminating the need for such expensive and potentially harmful sterilization methods.

The researchers engineered microbes, such as Escherichia coli, with the ability to extract nitrogen and phosphorous — two vital nutrients needed for growth — from unconventional sources that could be added to the fermentation vessels, according to Gregory Stephanopoulos, the Willard Henry Dow Professor of Chemical Engineering and Biotechnology at MIT, and Joe Shaw, senior director of research and development at Novogy, who led the research.

What’s more, because the engineered strains only possess this advantage when they are fed these unconventional chemicals, the chances of them escaping and growing in an uncontrolled manner outside of the plant in a natural environment are extremely low.

“We created microbes that can utilize some xenobiotic compounds that contain nitrogen, such as melamine,” Stephanopoulos says. Melamine is a xenobiotic, or artificial, chemical that contains 67 percent nitrogen by weight.

Conventional biofermentation refineries typically use ammonium to supply microbes with a source of nitrogen. But contaminating organisms, such as Lactobacilli, can also extract nitrogen from ammonium, allowing them to grow and compete with the producer microorganisms.

In contrast, these organisms do not have the genetic pathways needed to utilize melamine as a nitrogen source, says Stephanopoulos.

“They need that special pathway to be able to utilize melamine, and if they don’t have it they cannot incorporate nitrogen, so they cannot grow,” he says.

The researchers engineered E. coli with a synthetic six-step pathway that allows it to express enzymes needed to convert melamine to ammonia and carbon dioxide, in a strategy they have dubbed ROBUST (Robust Operation By Utilization of Substrate Technology).

When they experimented with a mixed culture of the engineered E. coli strain and a naturally occurring strain, they found the engineered type rapidly outcompeted the control, when fed on melamine.

They then investigated engineering the yeast Saccharomyces cerevisiae to express a gene that allowed it to convert the nitrile-containing chemical cyanamide into urea, from which it could obtain nitrogen.

The engineered strain was then able to grow with cyanamide as its only nitrogen source.

Finally, the researchers engineered both S. cerevisiae and the yeast Yarrowia lipolytica to use potassium phosphite as a source of phosphorous.

Like the engineered E. coli strain, both the engineered yeasts were able to outcompete naturally occurring strains when fed on these chemicals.

“So by engineering the strains to make them capable of utilizing these unconventional sources of phosphorous and nitrogen, we give them an advantage that allows them to outcompete any other microbes that may invade the fermenter without sterilization,” Stephanopoulos says.

The microbes were tested successfully on a variety of biomass feedstocks, including corn mash, cellulosic hydrolysate, and sugar cane, where they demonstrated no loss of productivity when compared to naturally occurring strains.

The paper provides a novel approach to allow companies to select for their productive microbes and select against contaminants, according to Jeff Lievense, a senior engineering fellow at the San Diego-based biotechnology company Genomatica who was not involved in the research.

“In theory you could operate a fermentation plant with much less expensive equipment and lower associated operating costs,” Lievense says. “I would say you could cut the capital and capital-related costs [of fermentation] in half, and for very large-volume chemicals, that kind of saving is very significant,” he says.

The ROBUST strategy is now ready for industrial evaluation, Shaw says. The technique was developed with Novogy researchers, who have tested the engineered strains at laboratory scale and trials with 1,000-liter fermentation vessels, and with Felix Lam of the MIT Whitehead Institute for Biomedical Research, who led the cellulosic hydrosylate testing.

Novogy now hopes to use the technology in its own advanced biofuel and biochemical production, and is also interested in licensing it for use by other manufacturers, Shaw says.

Kim Cobb, a marine scientist at the Georgia Institute of Technology, expected the coral to be damaged when she plunged into the deep blue waters off Kiritimati Island, a remote atoll near the center of the Pacific Ocean. Still, she was stunned by what she saw as she descended some 30 feet to the rim of a coral outcropping.

“The entire reef is covered with a red-brown fuzz,” Dr. Cobb said when she returned to the surface after her recent dive. “It is otherworldly. It is algae that has grown over dead coral. It was devastating.”

The damage off Kiritimati is part of a mass bleaching of coral reefs around the world, only the third on record and possibly the worst ever. Scientists believe that heat stress from multiple weather events including the latest severe El Niño, compounded by climate change, has threatened more than a third of Earth’s coral reefs. Many may not recover.

Coral reefs are the crucial incubators of the ocean’s ecosystem, providing food and shelter to a quarter of all marine species, and they support fish stocks that feed more than one billion people. They are made up of millions of tiny animals, called polyps, that form symbiotic relationships with algae, which in turn capture sunlight and carbon dioxide to make sugars that feed the polyps.

An estimated 30 million small-scale fishermen and women depend on reefs for their livelihoods, more than one million in the Philippines alone. In Indonesia, fish supported by the reefs provide the primary source of protein.

“This is a huge, looming planetary crisis, and we are sticking our heads in the sand about it,” said Justin Marshall, the director of CoralWatch at Australia’s University of Queensland.

Bleaching occurs when high heat and bright sunshine cause the metabolism of the algae — which give coral reefs their brilliant colors and energy — to speed out of control, and they start creating toxins. The polyps recoil. If temperatures drop, the corals can recover, but denuded ones remain vulnerable to disease. When heat stress continues, they starve to death.

Damaged or dying reefs have been found from Réunion, off the coast of Madagascar, to East Flores, Indonesia, and from Guam and Hawaii in the Pacific to the Florida Keys in the Atlantic.

The largest bleaching, at Australia’s Great Barrier Reef, was confirmed last month. In a survey of 520 individual reefs that make up the Great Barrier Reef’s northern section, scientists from Australia’s National Coral Bleaching Task Force found only four with no signs of bleaching. Some 620 miles of reef, much of it previously in pristine condition, had suffered significant bleaching.

In follow-up surveys, scientists diving on the reef said half the coral they had seen had died. Terry Hughes, the director of the Center of Excellence for Coral Reef Studies at James Cook University in Queensland, who took part in the survey, warned that even more would succumb if the water did not cool soon.

Toxins from harmful algae are present in Alaskan marine food webs in high enough concentrations to be detected in marine mammals such as whales, walruses, sea lions, seals, porpoises and sea otters, according to new research from NOAA and its federal, state, local and academic partners.

The findings, reported online today in the journal Harmful Algae, document a major northward expansion of the areas along the Pacific Coast where marine mammals are known to be exposed to algal toxins. Since 1998, algal toxin poisoning has been a common occurrence in California sea lions in Central California.

Certain drought-stressed wheat cultivars perform better when their roots are in symbiosis with beneficial fungi

Scientists at Aarhus University have discovered that fungi associated with plant roots may improve growth and yield of drought-stressed wheat.

Water scarcity has a negative impact on wheat production. As a consequence of exposure to drought, crops show poorer growth and lower yield. This is a serious problem as the predicted increase in frequency of extreme climate episodes will lead to multiple drought conditions during crop growth which in turn will reduce the yield of wheat, one of the world’s most important foods.

The UN Intergovernmental Panel on Climate Change predicts that drought stress in crops will become increasingly serious in the future. Globally, wheat yield is only 30-60 percent of its potential.

Fungi may help

A specific group of useful fungi – the so-called arbuscular mycorrhizal fungi (AM fungi) – may be able to help alleviate drought stress in wheat. These fungi live in a symbiotic relationship with plant roots. Recent research from Aarhus University demonstrates that the fungi can improve growth and yield in some wheat varieties under drought stress.

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